Organ arrest, protection and preservation

ABSTRACT

The present invention relates to a method for arresting, protecting and/or preserving an organ which includes administering effective amounts of (i) potassium channel opener or agonist and/or an adenosine receptor agonist and (ii) local anaesthetic to a subject in need thereof. The present invention also relates to a method for arresting, protecting and/or preserving an organ which comprises adding a composition which includes effective amounts of (i) a potassium channel opener or agonist and/or an adenosine receptor agonist and (ii) a local anaesthetic to the organ. The present invention further provides a pharmaceutical or veterinary composition which includes effective amounts of (i) a potassium channel opener or agonist and/or an adenosine receptor agonist and (ii) a local anaesthetic.

This application is a continuation of U.S. Ser. No. 09/937,181, nowissued, filed on Jan. 10, 2002, now U.S. Pat. No. 6,955,814, which is aU.S. National Phase of International Application No. PCT/AU00/00226,International filing date of Mar. 23, 2000, and which claims priority toAustralian Application No. PP9414 filed on Mar. 23, 1999, and AustralianApplication No. PQ4199 filed on Nov. 23, 1999, each of which isincorporated herein by reference.

The present invention relates to a method and pharmaceutical orveterinary composition for arresting, protecting and/or preservingorgans, in particular the heart during open-heart surgery,cardiovascular diagnosis or therapeutic intervention.

There are over 20,000 open-heart surgery operations each year inAustralia, over 800,000 in the United States and about 1,000,000 inEurope. Of those requiring open-heart surgery, about 1.2% areneonates/infants primarily as a consequence of congenital heart disease.

The heart may be arrested for up to 3 hours during open-heart surgery.High potassium cardioplegia (in excess of 15–20 mM) has been the basisof myocardial arrest and protection for over 40 years. Currently themajority of solutions used contain high potassium including the widelyused St Thomas No. 2 Hospital Solution which generally contains 110 mMNaCl, 16 mM KCl, 16 mM MgCl₂, 1.2 mM CaCl₂ and 10 mM NaHCO₃ and has a pHof about 7.8. High potassium solutions usually lead to a membranedepolarisation from about −80 to −50 mV. Notwithstanding hyperkalemicsolutions providing acceptable clinical outcomes, recent evidencesuggests that progressive potassium induced depolarisation leads toionic and metabolic imbalances that may be linked to myocardialstunning, ventricular arrhythmias, ischaemic injury, endothelial cellswelling, microvascular damage, cell death and loss of pump functionduring the reperfusion period. Infant hearts are even more prone todamage with cardioplegic arrest from high potassium than adult hearts.The major ion imbalances postulated are linked to an increased sodiuminflux which in turn activates the Na⁺/Ca²⁺ exchangers leading to a risein intracellular Ca²⁺. Compensatory activation of Na⁺ and Ca²⁺ ion pumpsthen occur, which activate anaerobic metabolism to replenish ATP with aconcomitant increase in tissue lactate and fall in tissue pH. Freeradical generation and oxidative stress have also been implicated inpotassium arrest and partially reversed by the administration ofantioxidants. In some cases, high potassium induced ischaemia has beenreported to have damaged smooth muscle and endothelial function.

In an attempt to minimise ischaemic damage during cardioplegic arrest,an increasing number of experimental studies have employed potassiumchannel openers instead of high potassium. Cardioprotection usingnicorandil, aprikalim or pinacidil is believed to be linked to theopening of the potassium channel which leads to a hyperpolarised state,a shortening of the action potential and decreasing Ca²⁺ influx into thecell. One shortfall however is that the heart takes the same time orlonger to recover with no improvement in function than with highpotassium cardioplegic solutions. Another limitation is that pinacidilrequires a carrier due to its low solubility in aqueous solutions. Thecarrier routinely used is dimethyl sulphoxide (DMSO) which iscontroversial when used in animal or human therapy.

Most investigators, including those who advocate using potassium channelopeners, believe that as soon as blood flow is halted and the arrestsolution administered, ischaemia occurs and progressively increases withtime. To reduce the likelihood of damage, we sought a cardioplegicsolution that would place the heart in a reversible hypometabolic stateanalogous to the tissues of a hibernating turtle, a hummingbird intorpor or an aestivating desert frog. When these animals drop theirmetabolic rate (some by over 90%), their tissues do not becomeprogressively ischaemic but remain in a down-regulated steady statewhere supply and demand are matched. An ideal cardioplegic solutionshould produce a readily reversible, rapid electrochemical arrest withminimal tissue ischaemia. The heart should accumulate low tissuelactate, utilise little glycogen, show minimal changes in high-energyphosphates, cytosolic redox (NAD/NADH) and the bioenergeticphosphorylation (ATP/ADP Pi) ratio and free energy of ATP. There shouldbe little or no change in cytosolic pH or free magnesium, minimal watershifts between the intracellular and extracellular phases, and no majorultrastructural damage to organelles such as the mitochondria. The idealcardioplegic solution should produce 100% functional recovery with noventricular arrhythmia, cytosolic calcium overload or other pumpabnormalities. There is no cardioplegic solution currently availablewhich fulfils all these requirements. We have now found that the heartcan be better protected during arrest and recovery by using thepotassium channel opener adenosine and the local anaesthetic lignocaine.

The action of adenosine is controversial. Adenosine has been shown toincrease coronary blood flow, hyperpolarise the cell membrane and act asa preconditioning agent via the ATP-sensitive potassium channel andadenosine related pathways including adenosine receptors notably the A1receptor. Adenosine is also known to improve myocardial recovery as anadjunct to high potassium cardioplegia. Furthermore, adenosine can beused as a pretreatment (whether or not it is present in the arrestingsolution) to reduce lethal injury. In one study, adenosine was shown torival potassium arrest solutions and more recently in bloodcardioplegia, it prevented post-ischaemic dysfunction in ischaemicallyinjured hearts. Adenosine is sometimes added as an adjunct to potassiumcardioplegia.

Lignocaine is a local anaesthetic which blocks sodium fast channels andhas antiarrhythmatic properties by reducing the magnitude of inwardsodium current. The accompanying shortening of the action potential isthought to directly reduce calcium entry into the cell via Ca²⁺selective channels and Na⁺/Ca²⁺ exchange. Recent reports also implicatelignocaine with the scavenging of free radicals such as hydroxyl andsinglet oxygen in the heart during reperfusion. Associated with thisscavenging function, lignocaine may also inhibit phospholipase activityand minimise membrane degradation during ischaemia. Lignocaine has alsobeen shown to have a myocardial protective effect and in one study wasfound to be superior to high potassium solutions. However, ourexperiments show that lignocaine alone at 0.5, 1.0 and 1.5 mM gavehighly variable functional recoveries using the isolated working ratheart.

According to one aspect of the present invention there is provided amethod for arresting, protecting and/or preserving an organ whichincludes administering effective amounts of (i) a potassium channelopener or agonist and/or an adenosine receptor agonist and (ii) a localanaesthetic to a subject in need thereof.

According to another aspect of the present invention there is providedthe use of (i) a potassium channel opener or agonist and/or an adenosinereceptor agonist and (ii) a local anaesthetic in the manufacture of amedicament for arresting, protecting and/or preserving an organ.

The present invention also provides (i) a potassium channel opener oragonist and/or an adenosine receptor agonist and (ii) a localanaesthetic for use in arresting, protecting and/or preserving an organ.

According to a further aspect of the present invention there is provideda pharmaceutical or veterinary composition which includes effectiveamounts of (i) a potassium channel opener or agonist and/or an adenosinereceptor agonist and (ii) a local anaesthetic.

While the present invention is particularly advantageous in arresting,protecting and/or preserving an organ while it is intact in the body ofthe subject, it will be appreciated that it may also be used to arrest,protect and/or preserve isolated organs.

Thus, the present invention still further provides a method forarresting, protecting and/or preserving an organ which includes adding acomposition which includes effective amounts of (i) a potassium channelopener or agonist and/or an adenosine receptor agonist and (ii) a localanaesthetic to the organ.

The term “adding” is used herein in its broadest sense to refer to anymethods of exposing the organ to the composition of the presentinvention, for example, bathing, perfusing or pumping via variousroutes.

The term “organ” is used herein in its broadest sense and refers to anypart of the body exercising a specific function including tissues andcells or parts thereof, for example, cell lines or organellepreparations. Other examples include circulatory organs such as theheart, respiratory organs such as the lungs, urinary organs such as thekidneys or bladder, digestive organs such as the stomach, liver,pancreas or spleen; reproductive organs such as the scrotum, testis,ovaries or uterus, neurological organs such as the brain, germ cellssuch as spermatozoa or ovum and somatic cells such as skin cells, heartcells i.e., myocytes, nerve cells, brain cells or kidney cells.

The method of the present invention is particularly useful in arresting,protecting and/or preserving the heart during open-heart surgeryincluding heart transplants. Other applications include reducing heartdamage before, during or following cardiovascular intervention which mayinclude a heart attack, angioplasty or angiography. For example, thecomposition could be administered to subjects who have suffered or aredeveloping a heart attack and used at the time of administration ofblood clot-busting drugs such as streptokinase. As the clot isdissolved, the presence of the composition may protect the heart fromfurther injury such as reperfusion injury. The composition may beparticularly effective as a cardioprotectant in those portions of theheart that have been starved of normal flow, nutrients and/or oxygen fordifferent periods of time. For example, the composition may be used totreat heart ischaemia which could be pre-existing or induced bycardiovascular intervention.

Thus, the present invention also provides a cardioplegic orcardioprotectant composition which includes effective amounts of (i) apotassium channel opener or agonist and/or an adenosine receptor agonistand (ii) a local anaesthetic.

The potassium channel openers or agonists may be selected fromnicorandil, diazoxide, minoxidil, pinicadil, aprikalim, cromokulim,NS-1619(1,3-dihydro-1-[2-hydroxy5(trifluoromethyl)phenyl]5-(trifluoromethyl)2-H-benimidazol-one),

amlodipine, Bay K 8644(L-type)(1,4-dihydro-26-dimethyl-5-nitro-4[2(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid (methyl ester)), bepridil HCl (L-type), calciseptine(L-type), omega-conotoxin GVIA (N-type), omega-conotoxin MVIIC (Q-type),cyproheptadine HCl, dantrolene sodium (Ca²⁺ release inhibitor),diltiazem HCl (L-type), filodipine, flunarizine HCl (Ca²⁺/Na⁺),fluspirilene (L-type), HA-1077 2HCl(1-(5 isoquinolinyl sulphonyl) homopiperazine.HCl), isradipine, loperamide HCl, manoalide (Ca²⁺ releaseinhibitor), nicardipine HCl (L-type), nifedipine (L-type), niguldipineHCl (L-type), nimodipine (L-type), nitrendipine (L-type), pimozide (L-and T-type), ruthenium red, ryanodine (SR channels), taicatoxin,verapamil HCl (L-type), methoxy-verapamil HCl (L-type), YS-035 HCl(L-type)N[2(3,4-dimethoxyphenyl)ethyl]-3,4-dimethoxy N-methyl benzeneethaneamine HCl) and AV blockers such as verapamil and adenosine. Itwill be appreciated that this list includes calcium antagonists aspotassium channel openers are indirect calcium antagonists.

Adenosine is particularly preferred as it is capable of opening thepotassium channel, hyperpolarising the cell, depressing metabolicfunction, possibly protecting endothelial cells, enhancingpreconditioning of tissue and protecting from ischaemia or damage.Adenosine is also an indirect calcium antagonist, vasodilator,antiarrhythmic, antiadrenergic, free radical scavenger, arresting agent,anti-inflammatory agent (attenuates neutrophil activation), metabolicagent and possible nitric oxide donor.

In a preferred embodiment, the present invention provides a method forarresting, protecting and/or preserving an organ which includesadministering effective amounts of adenosine and a local anaesthetic toa subject in need thereof.

Suitable adenosine receptor agonists include N⁶-cyclopentyladenosine(CPA), N-ethylcarboxamido adenosine (NECA),2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine(CGS-21680), 2-chloroadenosine,N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine,2-chloro-N-⁶-cyclopentyladenosine (CCPA),N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine(AB-MECA),([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentanecarboxamide (AMP579), N⁶-(R)-phenylisopropyladenosine (R-PLA),aminophenylethyladenosine 9APNEA) and cyclohexyladenosine (CHA).

The local anaesthetic can be selected from mexiletine, diphenylhydantoinprilocaine, procaine, mepivacaine and Class 1B antiarrhythmic agentssuch as lignocaine or derivatives thereof, for example, QX-314.Lignocaine is preferred as it is capable of acting as a localanaesthetic probably by blocking sodium fast channels, depressingmetabolic function, lowering free cytosolic calcium, protecting againstenzyme release from cells, possibly protecting endothelial cells andprotecting against myofilament damage. Lignocaine is also a free radicalscavenger and an antiarrhythmic.

As lignocaine acts by blocking sodium fast channels, it will beappreciated that other sodium channel blockers could be used instead ofor in combination with the local anaesthetic in the method andcomposition of the present invention. Examples of suitable sodiumchannel blockers include venoms such as tetrodotoxin.

Thus, in a particularly preferred embodiment there is provided a methodfor arresting, protecting and/or preserving an organ which includesadministering effective amounts of adenosine and lignocaine to a subjectin need thereof.

In another preferred embodiment there is provided a pharmaceutical orveterinary composition which includes effective amounts of adenosine andlignocaine.

For ease of reference, the “potassium channel opener or agonist and/oradenosine receptor agonist” and the “local anaesthetic” will hereinafterbe referred to as the “active ingredients”.

The method of the present invention involves the administration ofeffective amounts of the active ingredients for a time and underconditions sufficient for the organ to be arrested, protected and/orpreserved. The active ingredients may be administered separately,sequentially or simultaneously and in a single dose or series of doses.

The subject may be a human or an animal such as a livestock animal (e.g.sheep, cow or horse), laboratory test animal (e.g. mouse, rabbit orguinea pig) or a companion animal (e.g. dog or cat), particularly ananimal of economic importance.

It will be appreciated that the amounts of active ingredients present inthe composition will depend on the nature of the subject, the type oforgan being arrested, protected and/or preserved and the proposedapplication. In the case of a human subject requiring heart arrestduring open-heart surgery, the concentration of adenosine is preferablyabout 0.001 to about 20 mM, more preferably about 0.01 to about 10 mM,most preferably about 0.05 to about 5 mM and the concentration oflignocaine is preferably about 0.001 to about 20 mM, more preferablyabout 0.01 to about 10 mM, most preferably about 0.05 to about 5 mM. Inthe case of a human subject requiring treatment before, during orfollowing a heart attack or cardiovascular intervention, the preferredconcentrations of adenosine and lignocaine are set out in the tablebelow.

Site of Injection Type/Units Adenosine Lignocaine IntravenousInfusion 1. 0.001–10 1. 0.0001–20 mg/min/kg 2. 0.01–5 2. 0.01–10 3.0.01–1 3. 0.5–3 Intravenous Bolus 1. 0.0001–100 1. 0.001–1000 mg/kg 2.0.001–10 2. 0.01–100 Intracoronary Infusion 1. 0.0001–100 1. 005–50mg/min 2. 0.001–1 2. 0.005–5 (per heart) 3. 0.01–0.5 3. 0.05–2.5Intracoronary Bolus 1. 0.001–1000 1. 0.01–10,000 μg 2. 0.1–100 2. 1–1000(per heart) 3. 1–20 3. 10–200 1. = preferably 2. = more preferably 3. =most preferably

The active ingredients may be administered by any suitable routeincluding oral, implant, rectal, inhalation or insufflation (through themouth or nose), topical (including buccal and sublingual), vaginal andparenteral (including subcutaneous, intramuscular, intravenous,intrasternal and intradermal). Preferably, administration in open-heartsurgery or cardiovascular intervention applications will be achieved bymixing the active ingredients with the blood of the subject or subjectshaving a similar blood type. The active ingredients then enter thecoronary circulation generally via the aorta. Arrest may also beachieved by either continuous or intermittent delivery. For example,heart arrest may occur by either continuous or intermittent perfusionretrograde through the aorta in the Langendorff mode. However, it willbe appreciated that the preferred route will vary with the condition andage of the subject and the chosen active ingredients.

The composition of the present invention is highly beneficial at about15° C. to about 37° C., preferably about 20° C. to about 37° C., wherelonger arrest times using St Thomas No. 2 solution can only be achievedwhen the temperature is lowered, for example, down to about 4° C.

While it is possible for one or both of the active ingredients to beadministered alone, it is preferable to administer one or both of themtogether with one or more pharmaceutically acceptable carriers, diluentsadjuvants and/or excipients. Each carrier, diluent, adjuvant and/orexcipient must be pharmaceutically “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the subject. The compositions may conveniently be presentedin unit dosage form and may be prepared by methods well known in the artof pharmacy. Such methods include the step of bringing into associationthe active ingredient with the carrier which constitutes one or moreaccessory ingredients. Preferably, the compositions are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers, diluents, adjuvants and/or excipients.

The present invention also extends to a pharmaceutical or veterinarycomposition which includes the active ingredients and a pharmaceuticallyor veterinarily acceptable carrier, diluent, adjuvant and/or excipient.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredients; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredients may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methyl cellulose), fillers (e.g. lactose, microcyrstallinecellulose or calcium hydrogen phosphate), lubricants (e.g. magnesiumstearate, talc or silica), inert diluent, preservative, disintegrant(e.g. magnesium stearate, talc or silica), inert diluent, preservative,disintegrant (e.g. sodium starch glycollate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose), surface-active ordispersing agents. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

Liquid preparations for administration prior to arresting, protectingand/or preserving the organ may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g.almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid);and energy sources (e.g. carbohydrates such as glucose, fats such aspalmitate or amino acid).

Compositions suitable for topical administration in the mouth includelozenges comprising the active ingredients in a flavoured basis, usuallysucrose and acacia or tragacanth gum; pastilles comprising the activeingredients in an inert basis such as gelatin and glycerin, or sucroseand acacia gum; and mouthwashes comprising the active ingredients in asuitable liquid carrier.

For topical application for the skin, the active ingredients may be inthe form of a cream, ointment, jelly, solution or suspension.

For topical application to the eye, the active ingredients may be in theform of a solution or suspension in a suitable sterile aqueous ornon-aqueous vehicle. Additives, for instance buffers, preservativesincluding bactericidal and fungicidal agents, such as phenyl mercuricacetate or nitrate, benzalkonium chloride or chlorohexidine andthickening agents such as hypromellose may also be included.

The active ingredients may also be formulated as depot preparations.Such long acting formulations may be administered by implantation (e.g.subcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the active ingredients may be formulated with suitablepolymeric or hydrophobic materials (e.g. as an emulsion in an acceptableoil or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Compositions for rectal administration may be presented as a suppositryor retention enema with a suitable non-irritation excipient which issolid at ordinary temperatures but liquid at the rectal temperature andwill therefore melt in the rectum to release the active ingredients.Such excipients include cocoa butter or a salicylate.

For intranasal and pulmonary administration, the active ingredients maybe formulated as solutions or suspensions for administration via asuitable metered or unit dose device or alternatively as a powder mixwith a suitable carrier for administration using a suitable deliverydevice.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render thecomposition isotonic with the blood of the intended subject; and aqueousand non-aqueous sterile suspensions which may include suspending agentsand thickening agents. The compositions may be presented in unit-dose ormulti-dose sealed containers, for example, ampoules and vials, and maybe stored in a freeze-dried (lyophilised) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

When the composition is for verterinary use it may be prepared, forexample, by methods that are conventional in the art. Examples of suchveterinary compositions include those adapted for:

-   (a) oral administration, external application, for example drenches    (e.g. aqueous or non-aqueous solutions or suspensions); tablets or    boluses; powders, granules or pellets for admixture with feedstuffs;    pastes for application to the tongue;-   (b) parenteral administration for example by subcutaneous,    intramuscular or intravenous injection, e.g. as a sterile solution    or suspension; or (when appropriate) by intramammary injection where    a suspension or solution is introduced into the udder via the teat;-   (c) topical application, e.g. as a cream, ointment or spray applied    to the skin; or-   (d) intravaginally, e.g. as a pessary, cream or foam.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions of this invention may include otheragents conventional in the art having regard to the type of compositionin question, for example, those suitable for oral administration mayinclude such further agents as binders, sweeteners, thickeners,flavouring agents, disintegrating agents, coating agents, preservatives,lubricants and/or time delay agents.

Suitable sweeteners include sucrose, lactose, glucose, aspartame orsaccharin. Suitable disintegrating agents include corn starch,methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginicacid or agar. Suitable flavouring agents include peppermint oil, oil ofwintergreen, cherry, orange or raspberry flavouring. Suitable coatingagents include polymers or copolymers of acrylic acid and/or methacrylicacid and/or their esters, waxes, fatty alcohols, zein, shellac orgluten. Suitable preservatives include sodium benzoate, vitamin E,alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben orsodium bisulphite. Suitable lubricants include magnesium stearate,steric acid, sodium oleate, sodium chloride or talc. Suitable time delayagents include glyceryl monostearate or glyceryl distearate.

A preferred pharmaceutically acceptable carrier is a buffer having a pHof about 6 to about 9, preferably about 7, more preferably about 7.4and/or low concentrations of potassium, for example, up to about 10 mM,more preferably about 2 to about 8 mM, most preferably about 4 to about6 mM. Suitable buffers include Krebs-Henseleit which generally contains10 mM glucose, 117 mM NaCl, 5.9 mM KCl, 25 mM NaHCO₃, 1.2 mM NaH₂PO₄,1.12 mMCaCl₂ (free Ca²⁺=1.07 mM) and 0.512 mM MgCl₂ (free Mg²⁺=0.5 mM),St. Thomas No. 2 solution, Tyrodes solution which generally contains 10mM glucose, 126 mM NaCl, 5.4 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 0.33 mMNaH₂PO₄ and 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid], Fremes solution, Hartmanns solution which generallycontains 129 NaCl, 5 mM KCl, 2 mM CaCl₂ and 29 mM lactate andRingers-Lactate. One advantage of using low potassium is that it rendersthe present composition less injurous to the subject, in particularpediatric subjects such as neonates/infants. High potassium has beenlinked to an accumulation of calcium which may be associated withirregular heart beats during recovery, heart damage and cell swelling.Neonates/infants are even more susceptible than adults to high potassiumdamage during cardiac arrest. After surgery for defects aneonate/infant's heart may not return to normal for many days, sometimesrequiring intensive therapy or life support. It is also advantageous touse carriers having low concentrations of magnesium, such as, forexample up to about 2.5 mM, but it will be appreciated that highconcentrations of magnesium, for example up to about 20 mM, can be usedif desired without substantially effecting the activity of thecomposition.

In a further preferred embodiment the present invention provides apharmaceutical or veterinary composition which includes adenosine,lignocaine and a pharmaceutically acceptable carrier which contains upto about 10 mM potassium.

In a still further preferred embodiment, the present invention providesa pharmaceutical or veterinary composition which includes adenosine,lignocaine and Krebs-Henseleit buffer.

The composition may also advantageously be presented in the form of akit in which the active ingredients are held separately for separate,sequential or simultaneous administration.

It will be appreciated that the composition of the present invention mayalso include and/or be used in combination with known medicamentsdepending on the proposed application. For instance, medicaments whichsubstantially prevent the breakdown of adenosine in the blood such asnucleoside transport inhibitors, for example, dipyridamole could be usedas additives in the composition of the present invention. The half lifeof adenosine in the blood is about 10 seconds so the presence of amedicament to substantially prevent its breakdown will maximise theeffect of the composition of the present invention. Dipyridamole couldadvantageously be included in concentrations from about 0.1 nM to about10 mM and has major advantages with respect to cardioprotection.Dipyridamole may supplement the actions of adenosine by inhibitingadenosine transport which increases vasodilation. This could beparticularly important when the composition is administeredintermittently.

Other examples of medicaments include clot-busting drugs such asstreptokinase. As discussed earlier, the composition could beadministered at the time of administration of streptokinase in subjectswho have suffered or are developing a heart attack.

The invention will now be described with reference to the followingexamples. These examples are not to be construed as limiting in any way.

In the example, reference will be made to the accompanying drawings inwhich:

FIG. 1 is a graph of aortic flow vs time comparing hearts arrested using100 μM adenosine and 0.5 mM lignocaine in Krebs-Henseleit and St. ThomasHospital No. 2 solution;

FIG. 2 is six graphs showing heart, rate systolic pressure, aortic flow,coronary flow, MV02 and rate pressure product recovery from 30 minsintermittent ischaemia;

FIG. 3 is six graphs showing heart rate, systolic pressure, aortic flow,coronary flow, MV02 and rate pressure product recovery from 2 hrsintermittent ischaemia;

FIG. 4 is six graphs showing heart rate, systolic pressure, aortic flow,coronary flow, MV02 and rate pressure product recovery from 4 hrsintermittent ischaemia;

FIG. 5 is a bar graph providing a summary of the results of FIGS. 2 to4;

FIG. 6 is six graphs showing heart rate, systolic pressure, aortic flow,coronary flow, MV02 and rate pressure product recovery from 2 hrs ofintermittent ischaemia using neonate rat hearts;

FIG. 7 is four graphs showing 20 min ischaemia in rat heart in vivofollowing coronary artery ligation with no adenosine-lignocaineinfusion;

FIG. 8 is four graphs showing 20 min ischaemia in rat heart in vivofollowing coronary artery ligation when infused with adenosine (6.3mg/ml) and lignocaine (12.6 mg/ml) at 1 ml/hour/300 g rat;

FIG. 9 is a graph showing 30 min ischaemia in rat heart in vivofollowing coronary artery ligation when infused with adenosine (6.3mg/ml) and lignocaine (12.6 mg/ml) at 1 ml/hour/300 g rat;

FIG. 10 is four graphs showing 20 min ischaemia in rat heart in vivofollowing coronary artery ligation when infused with adenosine (3.15mg/ml) and lignocaine (12.6 mg/ml) at 1 ml/hour/300 g rat;

FIG. 11 is four graphs showing 30 min ischaemia in rat heart in vivofollowing coronary artery ligation when infused with adenosine (1.6mg/ml) and lignocaine (12.6 mg/ml) at 1 ml/hour/300 g rat;

FIG. 12 is a graph showing 30 min ischaemia in rat heart in vivofollowing coronary artery ligation when infused with adenosine (1.6mg/ml) and lignocaine (12.6 mg/ml) at 1 ml/hour/300 g rat;

FIG. 13 is two graphs showing the change in ATP and PCr versus time ofischaemia during a heart attack in vivo with and without the presence ofAL;

FIG. 14 is two graphs showing the change in lactate and myocardial pHversus time of ischaemia during a heart attack in vivo with and withoutthe presence of AL; and

FIG. 15 is two graphs showing the change in glycogen and rate pressureproduct versus time of ischaemia during a heart attack in vivo with andwithout the presence of AL.

In the examples, “AL” refers to compositions containing adenosine andlignocaine.

EXAMPLE 1

This example compares the effects of adenosine (100 μM) cardioplegiawith hyperkalemic St Thomas Hospital No. 2 solution (16 mM K⁺) onfunctional recovery after a period of global ischaemia using continuousperfusion.

Hearts from male 450 g Sprague-Dawley rats (n=19) were perfused for 30minutes in the working mode (preload 7.5 mmHg; afterload 100 mmHg) withKrebs-Henseleit pH 7.4 buffer at 37° C. Hearts were then arrested in aretrograde mode at a constant pressure of 70 mmHg with either (i) asolution containing 100 μM adenosine and 0.5 mM lignocaine in filteredKrebs-Henseleit (10 mM glucose, pH 7.6–7.8 @ 37° C.) (n=11) or (ii) St.Thomas No 2 solution (0.2 micron filter) (n=8). Following either 30minutes or 4 hrs of arrest, the hearts were switched back to normalantegrade perfusion with Krebs-Henseleit pH 7.4 @ 37° C. Heart rate,coronary flow, aortic flow, aortic pressure and oxygen consumption weremonitored. Statistical significance was assessed using a Student t-Test.

Results

Hearts arrested for 30 minutes using adenosine cardioplegia achievedquiescence in half the time compared to St. Thomas No. 2 solution (30 vs77 seconds, p<0.0001). During arrest under a constant perfusionpressure, coronary blood flow was 30% greater using adenosinecardioplegia (p<0.05). Faster recoveries were found in AL hearts inaortic pressure, aortic flow and cardiac output during reperfusion.After 5 min into reperfusion, the heart rate, aortic pressures, aorticflow, coronary flow, cardiac output and O₂ consumption were higher inthe AL hearts (Table 1). Higher aortic flows were also found at 15, 25and 35 min against a perfusion head of 100 mmHg (FIG. 1).

TABLE 1 Comparison between adenosine and lignocaine cardioplegia and StThomas No 2 Hospital solution after 30 min Normothermic ContinuousArrest in the working rat heart (37° C.) Adenosine and lignocaine ThomasNo 2 Solution (n = 11) (n = 12) Time to electromechanical arrest (sec)30 ± 2 sec 77 ± 6 sec Parameter Control 5 min Recovery % Control Control5 min Recovery % Control Heart (bpm) 292 ± 9 213 ± 8 73% 285 ± 14 150 ±35   53% Systolic pressure (mmHg) 122 ± 3 126 ± 4 103%  126 ± 3  88 ±14   70%* Diastolic Pressure (mmHg)  76 ± 1   74 ± 1.3 97% 78.5 ± 1.2 59± 8.5 75% Aortic flow (ml/min) 35.6 ± 3   24 ± 4 67% 31.5 ± 4.1 9.96 ±2.8    32%* Coronary flow (ml/min)  16.4 ± 0.7  13.6 ± 0.9 83%  17.4 ±0.74 10 ± 1.9 57% Cardiac Output (ml/min)  52 ± 3  37.2 ± 4.7 72% 50 ± 420 ± 4.5  40%* 0₂ consumption (μmol/min/g wet wt)  6.97 ± 0.28   5.39 ±0.38 77%  7.28 ± 0.30 4.14 ± 0.5   57% Control values are taken 5 minprior to the 30 min arrest protocol. *Significant P < 0.05 In terms offunctional parameters, 100 μM adenosine and 0.5 mM lignocainecardioplegia lead to shorter arrest times and an enhanced recoveryprofile compared to the St. Thomas Hospital No. 2 solution. The resultsfor hearts arrest for 4 hrs are shown in Table 2 below.

TABLE 2 Comparison of functional Recovery of S-D Rat Hearts After 30 minContinuous Cardioplegia With Adenosine/Lignocaine Cardioplegia or StThomas Hospital Solution No. 2 Heart Systolic Coronary Cardiac RatePressure Flow Aortic Flow Output MV02 n (bpm) (mmHg) (ml/min) (ml/min)(ml/min) (μmol/min/g) Arrest Stable Perfusion Period Adenosine + 7292.18 ± 8.82  122.38 ± 3.58 16.44 ± 1.07 35.66 ± 3.33   52 ± 2.73 6.97± 0.28 30 min Lignocaine % 100  100  100  100  100  100  CardoplegicCardioplegia Arrest St Thomas 10  2.85 ± 13.48 128.08 ± 3.14  17.4 ±0.74 31.53 ± 4.09 48.93 ± 4.15 7.28 ± 0.3  with Constant Hospital % 100 100  100  100  100  100  Perfusion Solution No 2 Delivered at 70 mmHgAfter 5 min Reperfusion Adenosine + 7 212.91 ± 7.62  126.09 ± 4.15  13.6± 0.92 23.64 ± 4.09 37.24 ± 4.73 5.39 ± 0.38 Lignocaine % 73 103  83 6672 77 Cardioplegia St Thomas 10 150.36 ± 34.45  88.08 ± 14.21 10.09 ±1.93  9.96 ± 2.83 20.06 ± 4.49 4.14 ± 0.5  Hospital % 53 70 57 32 40 57Solution No. 2 After 15 min Reperfusion Adenosine + 7 262.18 ± 10.36114.91 ± 4.18 12.55 ± 1.03 25.07 ± 3.08 37.82 ± 3.89 5.89 ± 0.32Lignocaine % 90 94 76 71 72 86 Cardioplegia St Thomas 10 257.09 ± 14.81118.82 ± 3.81 15.05 ± 1.24 16.18 ± 2.95 31.24 ± 3.73  6.1 ± 0.45Hospital % 90 94 86 51 64 84 Solution No. 2 After 25 min ReperfusionAdenosine + 7 253.54 ± 28.47  118.4 ± 3.58 14.08 ± 0.75 30.52 ± 2.7344.8 ± 3   8.06 ± 0.31 Lignocaine % 87 97 86 86 86 87 Cardioplegia StThomas 10 266.91 ± 15.16 118.09 ± 3.43 15.05 ± 1.04 23.11 ± 3.94 38.16 ±4.47  6.6 ± 0.48 Hospital % 94 94 86 73 78 91 Solution No. 2 After 35min Reperfusion Adenosine + 7 283.83 ± 11.74 118.88 ± 4.62  14.2 ± 0.6832.13 ± 2.94 46.33 ± 3.43 6.54 ± 0.09 Lignocaine % 97 97 88 90 89 94Cardioplegia St Thomas 10 271.27 ± 14.04 120.45 ± 3.11 15.38 ± 1.3725.35 ± 4.03 40.74 ± 4.4  6.74 ± 0.48 Hospital % 96 96 88 80 89 96Solution No. 2

EXAMPLE 2

Adult Wistar rats (350 g) were prepared using the method described inExample 2 and then subjected to intermittent perfusion as discussedbelow.

Intermittent retrograde perfusion was performed under a constantpressure head of 70 mmHg after hearts were switched back from theworking mode to the Lagendorff mode. After stabilisation, the heartswere arrested using 50 ml of either adenosine plus lignocainecardioplegia or St Thomas Hospital No 2 solution. The aorta was thencross-clamped and the heart left to sit arrested for 20 min (except in30 min intermittent arrest protocol), after which the clamp was releasedand 2 min of arrest solution delivered from a pressure head of 70 mmHg.The clamp was replaced and this procedure continued for up to 30 mins, 2hrs and 4 hrs at 37° C.

Intermittent cardioplegic delivery is the method commonly usedclinically in contrast to continuous perfusion in Example 1. DuringIntermittent arrest, the aorta of the subject is clamped and the arrestsolution administered. After a few minutes, the heart is arrested andcardioplegia delivery stopped. The heart remains motionless to permitsurgery. The arrest solution is administered again every 30 min for fewminutes to maintain the heart in the arrested state to preserve andprotect the heart muscle. Between these times, the heart muscle slowlybecomes ischaemic indicated by the production of lactate and fall inmuscle pH. For this reason, intermittent perfusion delivery is oftencalled intermittent ischaemic arrest. The results are shown in Tables 3to 7 below and FIGS. 2 to 5.

30 min Ischaemic Arrest at 37° C.

Table 3 and FIG. 2 show that A–L arrests in half the time of St Thomassolution 21s (n=7) vs 53s (n=10). All hearts returned function to thesame level following reperfusion (no significant difference betweengroups).

TABLE 3 Characteristics of Adult Heart 30 min Intermittent Arrest*Achieved by Adenosine/Lignocaine Cardioplegia and St Thomas HospitalSolution No. 2 *(2 min cardioplegia pulse after 15 min periods of aorticclamping) Adenosine/Lignocaine St Thomas Cardioplegia Hospital Solution(n = 7) No. 2 (n = 10) P Arrest Time (s) 21.43 ± 3.92 52.78 ± 5.65 p <0.01 Time to First 147.14 ± 14.95 133.67 ± 31.44 ns ContractionFollowing Reperfusion (s) Time to Recover 302.14 ± 21.87 309.44 ± 30.15ns 100 mmHg

TABLE 4 Comparson of functional Recovery of Rat Hearts after 30 minIntermittent Ischaemia* With Adenosine/Lignocaine Cardioplegia or StThomas Hospital Solution No 2 Heart Systolic Aortic Coronary CardiacRate Pressure Flow Flow Output RP Product MV02 n (bpm) (mmHg) (ml/min)(ml/min) (ml/min) (mmHg/min) (μmol/min/g) Arrest Stable Perfusion PeriodAdenosine + 7 245.38 ± 11.01 128.23 ± 2.83 34.33 ± 3.64 21.64 ± 2.0258.29 ± 4.63 31504 ± 1651 6.31 ± 0.65 30 min Lignocaine IschaemiaCardioplegia Arrest with St Thomas 10 276.74 ± 11.87 123.64 ± 1.30 32.78± 2.09 19.38 ± 1.62 55.36 ± 2.59 34090 ± 1111 5.97 ± 0.56 CardioplegiaHospital 100  100  100  100  100  100  100  Delivered Solution at 15 minNo 2 After 5 min Reperfusion Adenosine + 7 180.48 ± 26.83 132.79 ± 6.6522.06 ± 4.48 *22.15 ± 2.20  47.59 ± 2.70 24074 ± 3330 6.81 ± 0.97Lignocaine 74 104  64 102  82 76 108  Cardioplegia St Thomas 10 135.94 ±32.71  81.82 ± 15.94 19.04 ± 4.69 *13.48 ± 2.47  34.61 ± 6.96 23281 ±4069 5.02 ± 0.79 Hospital 49 58 70 63 68 84 Solution No 2 After 15 minReperfusion Adenosine + 7 225.31 ± 19.17 126.17 ± 2.88 29.21 ± 3.2017.14 ± 1.81 48.99 ± 3.76 28228 ± 2015 5.03 ± 0.49 Lignocaine 92 98 8579 84 90 80 Cardioplegia St Thomas 10 255.88 ± 9.69  121.56 ± 1.32 24.84± 2.36 17.00 ± 1.64 45.07 ± 2.47 31131 ± 1267 5.41 ± 0.53 Hospital 92 9876 88 81 91 91 Solution No 2 After 30 min Reperfusion Adenosine + 7236.94 ± 13.75 124.84 ± 2.61 29.60 ± 2.83 16.49 ± 1.51 49.09 ± 1.9529403 ± 1231 5.42 ± 0.70 Lignocaine 97 97 86 76 84 93 86 Cardioplegia StThomas 10 255.17 ± 12.29 122.16 ± 1.62 22.26 ± 3.32 17.08 ± 1.20 42.41 ±3.28 31154 ± 1464 5.26 ± 0.38 Hospital 92 99 68 88 77 91 88 Solution No2 After 60 min Reperfusion Adenosine + 7 244.97 ± 11.48 119.80 ± 2.9522.42 ± 3.48 15.52 ± 0.49 41.53 ± 2.78 29269 ± 1240 5.25 ± 0.55Lignocaine 100 93 65 72 71 93 83 Cardioplegia St Thomas 10 258.16 ±13.88 117.57 ± 1.68 17.01 ± 3.08 15.46 ± 1.21 35.89 ± 3.46 30392 ± 17275.08 ± 0.33 Hospital 93 95 52 80 65 89 85 Solution No 2 *StatisticallySignificant Difference Using Students TTEST (p < 0.05)2 hr Ischaemic Arrest at 37° C.

Table 5 and FIG. 3 show that A–L arrests in half the time of St Thomassolution 33s (n=7) vs 81s (n=8). 4 out of 8 hears arrested with StThomas did not recover. All A–L hearts survived (n=7). St Thomas heartswhich recovered (n=4) had 50–90% aortic flow, 70–120% heart rate and 90–100% systolic pressure. A–L hearts recovered 80% aortic flow, 95% heartrate and 95–100% systolic pressure.

TABLE 5 Characteristics of Adult Rat Heart 2 hr Ischaemic Arrest*Achieved by Adenosine/Lignocaine Cardioplegia and St Thomas HospitalSolution No. 2 *(2 min Cardioplegia pulse repeated after 20 min ofaortic clamping) Adenosine/ St Thomas Lignocaine Hospital n Cardioplegian Solution No. 2 p Arrest Time(s) 7 33 ± 5  8 81 ± 8  0.0003 Time toFirst 7 360 ± 19  4 260 ± 95  NS Contraction following Reperfusion(s)Time to Recover 7 541 ± 46  4 2400 ± 3261 NS 100 mmHg and Achieve Aorticflow(s) Percentage of Hearts 100 50 to Survive Reperfusion4 hr Ischaemic Arrest at 37° C.

Tables 6 and 7 and FIG. 4 show A–L arrests in half the time of St Thomassolution (26s (n=9) vs 78s (n=7)). 6 out of 7 hearts arrested with StThomas did not recover. All A–L hearts survived (n=9). The single StThomas heart which recovered had 40% aortic flow, 80% heart rate and 90%systolic pressure. A–L hearts recovered 70% aortic flow, 90% heart rateand 95–100% systolic pressure.

TABLE 6 Characteristics of Adult Rat Heart 4 hr Ischaemic Arrest*Achieved by Adenosine/Lignocaine Cardioplegia and St Thomas HospitalSolution No. 2 *(2 min cardiplegia pulse repeated after 20 min of aorticclamping) St Thomas Adenosine/ Hospital Lignocaine Solution CardioplegiaNo. 2 p Arrest Time(s) 26.44 ± 2.77 77.86 ± 10 <0.001 (n = 9) (n = 7)Time to First 401.67 390.00 Contraction following 28.48 (n = 1)Reperfusion(s) (n = 9) Time to Recover 549.22 480.00 100 mmHg andAchieve 40.68 (n = 1) Aortic flow(s) (n = 9) Percentage of Hearts to 10014 <0.0001 Survive Reperfusion (n = 9) (n = 1)

TABLE 7 Comparison of function Recover of Rat Hearts After 4 hrIntermittent Ischaemic Arrest with Adenosine/Lignocaine Cardioplegia orSt Thomas Hospital Solution No. 2 Heart Systolic Aortic Coronary CardiacMV02 Rate Pressure Flow Flow Output RP Product (μmol/ n (bpm) (mmHg)(ml/min) (ml/min) (ml/min) (mmHg/min) min/g) Arrest Stable PerfusionPeriod Adenosine + 9 275.33 ± 118.44 ± 36.47 ± 16.28 ± 53.88 ± 32338 ±6.71 ± 4 hr Ischaemic Lignocaine 12.91 3.50 1.65 1.03 1.73 1084 0.45Arrest with Cardioplegia 2 min St Thomas 7 259.21 ± 121.57 ± 41.23 ±16.03 ± 57.26 ± 31508 ± 7.64 ± Cardioplegia Hospital (n = 1) 12.84 2.424.18 1.26 5.30 1672 0.24 Delivered Solution 270 117.00 51 19.8 70.8315900 7.28 Every 20 min No. 2 After 15 min Reperfusion Adenosine + 9229.89 ± 110.89 ± 19.81 ± 13.92 ± 36.49 ± 25327 ± 5.94 ± Lignocaine16.10 1.86 3.56 1.53 4.13 1555 0.69 Cardioplegia % 83 94 54 86 68 78 89St Thomas 1 220.00 100 18.60 16.20 36.40 22000 5.303 Hospital % 81 85 3682 51 70 73 Solution No. 2 After 30 min Reperfusion Adenosine + 9239.444 ± 113.00 ± 24.62 ± 11.53 ± 39.44 ± 26684 ± 4.946 ± Lignocaine18.7165 3.07 2.917 1.001 4.259 1669 0.443 Cardioplegia % 87 95 68 71 7383 74 St Thomas 1 220 105.00 16.8 20.4 39.2 23100 5.303 Hospital % 81 9033 103 55 73 73 Solution No. 2 After 60 min Reperfusion Adenosine + 9249.22 ± 111.89 ± 25.58 ± 11.39 ± 40.63 ± 27570 ± 5.04 ± Lignocaine17.19 3.29 3.26 1.32 4.72 1577 0.49 Cardioplegia % 91 94 70 70 75 85 75St Thomas 1 250.00 102.00 14.40 18.00 34.40 25500 6.29 Hospital % 93 8728 91 49 81 86 Solution No. 2

FIG. 5 is a summary of the results of FIGS. 2 to 4 which shows heartsarrested with AL solution all survived after 30 min ischaemicintermittent arrest (n=7), 2 hrs intermittent arrest (n=7) and 4 hrs ofintermittent arrest (n=9). In contrast, while all hearts arrested withSt Thomas solution survived after 30 min (n=10), only 50% (4 out of 8tested) and 14% survived (1 out of 7) tested. In addition, two heartshave been arrested with AL successfully for 6 hrs (FIG. 5).

FIGS. 2 to 4 show the functional properties (heart rate, systolicpressure, aortic flow, coronary flow, oxygen consumption andrate-pressure product) during 60 min after 0.5 hr arrest (FIG. 2) 2 hrarrest (FIG. 3) and 4 hrs arrest (FIG. 4). In all cases, hearts arrestedwith AL solution had higher functional recovery parameters. After 0.5 hrarrest, these differences were not significant except for aortic flowrecover in hearts receiving AL arrest solution. Aortic flow against apressure head of 70 mmHg recovered to 90% of control values at 30 mincompared to 65% in St Thomas hearts. After 2 hr intermittent ischaemicarrest the differences in functional recover are more striking. In ALarrested hearts, heart rate and systolic pressure recovered to nearly100% of control values whereas St. Thomas hearts only recovered 40–50%.Aortic flow, coronary flow, oxygen consumption and rate-pressure productrecovered 80% and above the controls in AL hearts and only 20–40% in StThomas hearts. After 4 hr arrest the differences were even grater withonly 1 out of 7 St Thomas hearts recovering. All AL hearts recoveredafter 4 hr arrest with similar recovery functional profiles describedabove for 2 hr. It can be concluded that AL arrest provides superiorprotection during 2 and 4 hr arrest and recovery in adult hearts.

EXAMPLE 3

Neonatal/infant rat hearts (using 50–70 g 20 day old rats) were preparedusing the intermittent perfusion technique for 2 hr at 37° C. describedin Example 2 except the pressure head of delivery and afterload wasreduced to 50 mmHg. The results shown in Tables 8 and 9 below and FIG. 6show that A–L arrests in a third of the time of St Thomas solution 19s(n=7) vs 66s (n=7). 3 out of 7 hearts arrest with St Thomas did notrecover. All A–L hearts survived (n=7) with 80% aortic flow. The StThomas hearts which recovered averaged 80% aortic flow rate, but thiswas extremely variable.

All neonatal/infant hearts arrested with AL solution recovered after 2hr intermittent ischaemic arrest. Only 4 out o 7 hearts arrested with StThomas solution recovered after 2 hr intermittent ischaemic arrest. InAL arrested hearts, heart rate and systolic pressure recovered to90–100% of control values wherein St Thomas' hearts there was only50–60% recovery. Aortic flow, coronary flow and rate-pressure productrecovered to 80% and above the controls in AL hearts and only about 50%in St Thomas hearts. Oxygen consumption in the AL hearts was 70–85% ofcontrols and about 60% for the hearts arrested with St Thomas solution.It can be concluded that AL arrest provides superior protection during 2hr arrest and recover in neonatal/infant hearts.

TABLE 8 Characteristics of Neonatal Immature Rat Heart Arrest* Achievedby Adenosine/Lignocaine Cardioplegia and St Thomas Hospital No. 2 *(2min Cardioplegia pulse repeated after 20 min of aortic clamping).Reperfusion afterload of 50 mmHg. Adenosine/ St Thomas HospitalLignocaine Solution No. 2 p Arrest Time(s) 18.57 ± 3.72(7) 65.71* ±12.71(7) <0.05 Time to First 23.83 ± 3.03(7) 55.75* ± 12.97(4) <0.05Contraction Following Reperfusion(s) Time to Recover   165 ± 29.48(7)  270 ± 83.5(4) ns 50 mmHg Aortic flow(s) Percentage of 100(7)  57*(4)<0.05 Hearts to Survive Reperfusion Arrhythmia  14(7) 25(4) nsOccurrence (%) *Denotes Statistical Significance p < 0.05 using Studentst-test

TABLE 9 Comparison of functional Recovery of Immature Rat Hearts After 2hr Ischaemic* Arrest With Adenosine/Lignocaine Cardioplegia or St ThomasHospital Solution No. 2 *(2 min cardioplegia doses delivered between 20min periods of aortic clamping) Heart Coronary Cardiac Rate Aortic FlowFlow Output RP Product MV02 n (bpm) (ml/min) (ml/min) (ml/min)(mmHg/min) (μmol/min/g) Arrest Stable Perfusion Period Adenosine + 7261.98 ± 12.81 6.99 ± 1.30 4.80 ± 0.56 13.45* ± 1.16  16579 ± 853  4.60± 0.52 2 Hr Ischaemic Lignocaine Arrest with 2 min CardioplegiaCardioplegia St Thomas 7 239.96 ± 19.52 4.43 ± 1.05 4.29 ± 0.76 9.95* ±1.01 15515 ± 1149 5.39 ± 0.61 Delivered Every Hospital 20 min SolutionNo 2 After 15 min Reperfusion Adenosine + 7 230.94 ± 12.33 5.37 ± 1.634.03 ± 0.61 11.77 ± 1.84 14046 ± 1297 4.71 ± 0.33 LignocaineCardioplegia St Thomas 4 233.45 ± 36.2  3.78 ± 1.51 4.58 ± 1.2  10.87 ±1.99 13818 ± 3103 4.36 ± 0.67 Hospital Solution No. 2 After 30 minReperfusion Adenosine + 7 235.39 ± 8.99  6.67 ± 1.58 4.09 ± 0.68 12.45 ±1.71 14994 ± 709  4.43 ± 0.24 Lignocaine Cardioplegia St Thomas 4 228.55± 32.67 4.38 ± 1.68 4.00 ± 1.14 10.63 ± 1.97 13522 ± 2912 4.14 ± 0.69Hospital Solution No. 2 After 60 min Reperfusion Adenosine + 7 242.78 ±30.35   6 ± 1.66 3.75 ± 0.57 13.05 ± 1.55 13272 ± 2643 4.13 ± 0.62Lignocaine Cardioplegia St Thomas 4 234.48 ± 40.16 3.88 ± 1.41 3.58 ±0.92  9.68 ± 1.50 13910 ± 3262 4.02 ± 0.75 Hospital Solution No 2

EXAMPLE 4

Table 10 below shows that adenosine and lignocaine are effective in 1–2day old neonatal pig heart cardioplegia. (2 hours of 2 min pulses ofcardioplegia administered between 20 min periods of aortic clamping).

TABLE 10 Heart Rate Recovery n Arrest Time (s) (After 2 hr Arrest*) 1 875%

EXAMPLE 5

Male Wistar rats (250 g) were housed in a temperature andlight-controlled room. Food and water were provided freely until the daybefore the experiment when the food was withheld and the rats werefasted overnight. The rats were anaesthetised with an intraperitonealinjection of pentobarbital (60 mg kg⁻¹). Under anaesthesia, the ratswere implanted with cannulas in the femoral vein and artery foradenosine and lignocaine (AL) administration and blood pressuremeasurement, respectively. A tracheotomy was performed and the rats wereartificially ventilated with room air at 60 to 70 breaths/min. Thechests of the rats were cut open and the left anterior descending (LAD)coronary artery located. A piece of suture was placed underneath LAD.After a 20 min baseline period, LAD of the group of experimental ratswere ligated for 30 min and blood pressure and heart rate monitored.After 30 min of ischaemia, the ligature was released and the heartreperfused for 20 min. In the control rats, no AL was administered asshown in FIG. 7. In the AL infusion 3 rats were used at three differentdoses of adenosine:

-   (1) 6.3 mg/ml adenosine+12.6 mg/ml lignocaine infused at 1 ml/hr/300    g rat as shown in FIGS. 8 and 9;-   (2) 3.15 mg/ml adenosine+12.6 mg/ml lignocaine infused at 1    ml/hr/300 g rat as shown in FIG. 10; and-   (3) 1.6 mg/ml adenosine+12.6 mg/ml lignocaine infused at 1 ml/hr/300    g rat as shown in FIGS. 11 and 12.

Compared to rats with 30 min ischaemia (no AL infusion) it was foundthat AL protected the heart in a dose dependent manner with the greatestprotection occurring at the higher doses. As the dose of adenosine washalved, the protection was progressively lost. However, even in theworse case, the function of the heart was significantly better than withno AL alone. All hearts in rats receiving AL recovered in rate andpressure.

Summary of Adenosine and Lignocaine During a Heart Attack In Vivo

During a 30 min heart attack or myocardial infarction (MI) in the ratmodel, FIG. 7 shows that at 10 min blood pressure approaches zero andthe animal would be considered close to death. After 10 min, the heartrecovers and blood pressure increases and is highly erratic from theischaemic insult. This recovery is probably due to the recruitment ofcollateral circulation. In contrast, when a solution of adenosine andlignocaine is infused into the rat 5 min before occluding the coronaryartery, no such fall in blood pressure is seen at 10 min (FIG. 8). Wherethe animal without receiving AL solution nearly died at 10 min, in thepresence of AL solution the heart lowers its rate of contraction andmisses only a few beats. Noteworthy, there was no irregular beating ofthe heart at 20 min of ischaemia. All hearts recovered to full functionafter AL infusion was stopped (FIG. 9). It can be concluded that theheart in the presence of AL solution was dramatically protected againsta profound ischaemic insult elicited by occluding the coronary artery.The protective effect of the AL solution on the heart was related to thedose of adenosine. If the amount of adenosine was halved but the amountof lignocaine remained constant, more variability at 10 min and 20 isseen (FIG. 10). If the amount of adenosine was halved again, theprotection was reduced further. In all cases however, AL infused ratsfully recovered haemodynamic function based on blood pressure and heartrate (FIG. 12).

Two groups of rats undergoing a heart attack with and without a solutionof AL were placed in a nuclear magnetic resonance (NMR) spectrometer andthe metabolic data is shown in FIGS. 13 to 15. NMR non-invasivelymeasures the changes in adenosine-triphosphate (ATP), phosphocreatine(PCr) and pH during 30 min of coronary artery occlusion. In a separateexperiment on the bench, hearts were freeze-clamped at liquid nitrogentemperatures and glycogen and lactate were measured using routineenzymatic methods on neutralised tissue acid-extracts using aspectrophotometer. Major significant differences (P<0.05) were seen inthe hearts receiving AL solution during coronary artery occlusion. ATPremained between 90–100% of the control values in AL hearts compared to60% in hearts receiving no AL (FIG. 13). The same was shown for thehigh-energy phosphate store PCr, although greater percentage falls wereshown in hearts with no AL (down to as low as 20% of pre-occlusionvalues) (FIG. 14). In hearts receiving AL over the ischaemic periodlactate, an end-product of anaerobic metabolism, increased 5-foldwhereas lactate in hearts without AL increased over 20-fold (FIG. 15).This was also supported by measuring the myocardial cell pH; greaterdecreases in pH (more acid) are seen in hearts not receiving ALsolution. Noteworthy, in the first 10 min the pH fell only slight in ALhearts indicating that the myocardial cells in the presence of AL weremore aerobic supported by the lower tissue lactate levels. The fuelglycogen was used in similar amounts by hearts with and without AL inthe first 10 min but remained at about 60–70% of the pre-occlusionvalues in AL hearts compared to ischaemic hearts alone. It can beconcluded from the metabolic data that coronary-occluded heartsreceiving AL remained more aerobic than those hearts not receiving AL.Glycogen was a major source of fuel for each heart but the AL heartspreferentially regenerated their ATP from mitochondrial oxidativephosphorylation not from lactate production. This is wholly consistentwith the functional data discussed above from changes in blood pressureand heart rate.

EXAMPLE 6

Arrest solutions were made with 200 μM and 50 μm of the localanaesthetics prilocaine, procaine and mepivacaine in Krebs-Henseleithaving 10 mM glucose at pH 7.4. The results shown in Table 11 below arefor 30 min constant perfusion of cardioplegia at 70 mmHg.

TABLE 11 Adenosine + Adenosine + Adenosine + PRILOCAINE PROCAINEMEPIVACAINE ARREST TIME 13 s 21 s 10.5 s 1st BEAT 1:13 1:45 0:36 AORTICFLOW 3:12 3:35 3:40 RECOVERY 5 min AF % 67% 58% 39%

EXAMPLE 7

Arrest solutions were made with pinacidil dissolved in 0.05%dimethysulfoxide (DMSO) (200 μM) the local anaesthetics prilocaine,procaine, mepivacaine and lignocaine in Krebs-Henseleit solution. Asshown in Table 12 below, pinacidil was found to be not as effective asadenosine.

TABLE 12 Pinacidil + PRILOCAINE Pinacidil + PROCAINE Pinacidil +MEPIVACAINE Pinacidil + LIGNOCAINE Arrest Time 1:28 4:22 s 0:41 1:491^(st) Beat 2:15 1:20  0:56 2:30 Aortic Flow 8:10 4:50  6:55 4:45Recovery  5 min AF %  0% 25%  0% 70% 15 min AF % 38% 57% 36% 71%

EXAMPLE 8

The addition of the ATP-potassium channel blocker, glibenclamide (20 μM)and adenosine and lignocaine, delayed arrest times more than threefoldfrom 26 sec (AL) to 76–120 sec (ALG) (n=2). Furthermore the slowerrecovery times and lower aortic flow (42–53%) in the presence ofglibenclamide shows the importance of opening the KATP channels as amode of arrest and protection afforded by AL. It can be concluded fromthese results that the ATP-potassium channel is an important targeteliciting the arrest response from adenosine and lignocaine.

TABLE 13 A/L + 20 μM Glibenclamide A/L Alone (n = 2) (n = 5) Arrest Time  76–120 s 26.s 1st Beat 2:45–2:55 (min:s) 1 min:37 s Aortic Flow5:00–7:30 (min:s) 3 min:51 s Recovery Time 5 min AF %   42–53% 84%

1. A composition comprising: a pharmaceutically acceptable carrier; acompound chosen from a potassium channel opener, a potassium channelagonist and an adenosine receptor agonist; and a local anesthetic;wherein the compound and the local anesthetic are present in thecomposition in an amount sufficient to protect an organ.
 2. Thecomposition of claim 1, wherein the compound is a potassium channelopener or potassium channel agonist selected from nicorandil, diazoxide,minoxidil, pinacidil, aprikalim, cromokulim, NS-1619(1,3-dihydro-1-[2-hydroxy5(trifluoromethyl)phenyl]5-(trifluoromethyl)2-H-benimidazol-one),amlodipine, Bay K 8644(L-type)(1,4-dihydro-26-dimethyl-5-nitro-4[2(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid (methyl ester)), bepridil HCl (L-type), calciseptine(L-type), omega-conotoxin GVIA (N-type), omega-conotoxin MVHIIC(Q-type), cyproheptadine HGl, filodipine, fluspirilene (L-type), HA-10772HGl(1-(5 isoquinolinyl sulphonyl) homo piperazine.HGl), isradipine,loperamide HCl, pimozide (L- and T-type), ruthenium red, ryanodine (SRchannels), taicatoxin, verapamil HCl (L-type), methoxy-verapamil HCl(L-type), YS-035 HCl(L-type)N[2(3,4-dimethoxyphenyl)ethyl]-3,4-dimethoxy N-methyl benzeneethaneamine HCl) and AV blockers.
 3. The composition of claim 1, whereinthe compound is an adenosine receptor agonist selected fromN⁶-cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA),2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine(CGS-21680),2-chloroadenosine,N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine,2-chloro-N⁶-cyclopentyladenosine (CCPA),N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine(AB-MECA), ([IS-[1a, 2b, 3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentanecarboxamide (AMP579, N⁶-(R)-phenylisopropyladenosine (R-PLA),aminophenylethyladenosine [9](APNEA) and cyclohexyladenosine (CHA). 4.The composition of claim 1, wherein the local anesthetic is selectedfrom mexiletine, diphenylhydantoin, prilocaine, procaine, mepivicaineand Class 1B antiarrhythmic agents.
 5. The composition of claim 4,wherein the Class 1B antiarrhythmic agents is lignocaine.
 6. Thecomposition of claim 1, wherein the pharmaceutically acceptable carriercomprises a buffer which maintains the pH of the composition in therange from about 6 to about
 9. 7. The composition according to claim 1,wherein the pharmaceutically acceptable carrier includes potassium at aconcentration of less than about 10 mM.
 8. The composition of claim 1further including magnesium.
 9. The composition of claim 1 furtherincluding magnesium up to 2.5 mM.
 10. The composition of claim 1 whereinthe organ is a heart.
 11. Method of protecting an organ including thestep of contacting the organ with a composition according to claim 1.12. The method of claim 11 wherein the composition is at a temperaturebetween about 15° C. to about 37° C.
 13. The method of claim 11 whereinthe organ is a heart.
 14. The method of claim 11 wherein the organ is aheart of a patient with heart ischaemia.
 15. The method of claim 11wherein the compound is adenosine and the local anaesthetic islignocaine.
 16. The method of claim 11 wherein the organ is a heart andthe composition is administered to a subject who has suffered or isdeveloping a heart attack.
 17. A method of protecting an organcomprising contacting the organ with a composition according to claim 8.18. A method of protecting an organ comprising contacting the organ witha composition according to claim 8 to treat heart ischaemia.
 19. Amethod of reducing heart damage before, during or followingcardiovascular intervention comprising administering the composition ofclaim 8 to a subject who has suffered or is developing a heart attack.20. A composition comprising: a pharmaceutically acceptable carrier; acompound chosen from a potassium channel opener, a potassium channelagonist and an adenosine receptor agonist; and a local anesthetic;wherein the compound and the local anesthetic are present in thecomposition in an amount sufficient to preserve an organ.
 21. Thecomposition of claim 20 wherein the compound is a potassium channelopener or potassium channel agonist selected from nicorandil, diazoxide,minoxidil, pinacidil, aprikalim, cromokulim, NS-1619(1,3-dihydro-1-[2-hydroxy5(trifluoromethyl)phenyl]5-(trifluoromethyl)2-H-benimidazol-one),amlodipine, Bay K8644(L-type)(1,4-dihydro-26-dimethyl-5-nitro-4[2(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid (methyl ester)), bepridil HCl (L-type), calciseptine(L-type), omega-conotoxin GVIA (N-type), omega-conotoxin MVIIC (Q-type),cyproheptadine HGl, filodipine, fluspirilene (L-type), HA-10772HCl(1-(5isoquinolinyl sulphonyl) homo piperazine.HCl), isradipine,loperamide HGl, pimozide (L- and T-type), ruthenium red, ryanodine (SRchannels), taicatoxin, verapamil HCl (L-type), methoxy-verapamil HCl(L-type), YS-035 HCl(L-type)N[2(3,4-dimethoxyphenyl)ethyl]-3,4-dimethoxy N-methyl benzeneethaneamine HCl) and AV blockers.
 22. The composition of claim 20,wherein the compound is an adenosine receptor agonist selected fromN⁶-cyclopentyladenosine (CPA), N-ethylcarboxamido adenosine (NECA),2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine(CGS-21680),2-chloroadenosine,N⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine,2-chloro-N⁶-cyclopentyladenosine (CCPA),N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-robofuranosyl]-adenine(AB-MECA), ([IS-[1a, 2b, 3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentanecarboxamide (AMP579, N⁶-(R)-phenylisopropyladenosine (R-PLA),aminophenylethyladenosine [9](APNEA) and cyclohexyladenosine (CHA). 23.The composition of claim 20, wherein the local anesthetic is selectedfrom mexiletine, diphenylhydantoin, prilocaine, procaine, mepivacaineand Class B antiarrhythmic agents.
 24. The composition of claim 23,wherein the Class 1B antiarrhythmic agents is lignocaine.
 25. Thecomposition of claim 20, wherein the composition is a cardioplegic orcardioprotectant composition.
 26. The composition of claim 20, whereinthe pharmaceutically acceptable carrier comprises a buffer whichmaintains the pH of the composition in the range from about 6 to about9.
 27. The composition of claim 20, wherein the pharmaceuticallyacceptable carrier includes potassium at a concentration of less thanabout 10 mM.
 28. The composition of claim 20 further includingmagnesium.
 29. The composition of claim 20 further including magnesiumup to 2.5 mM.
 30. The composition of claim 20 wherein the organ is aheart.
 31. Method of preserving an organ including the step ofcontacting the organ with a composition according to claim
 20. 32. Themethod of claim 31 wherein the composition is at a temperature betweenabout 15° C. to about 37° C.
 33. The method of claim 31 wherein thecompound is adenosine and the local anaesthetic is lignocaine.
 34. Acomposition comprising: a pharmaceutically acceptable carrier;adenosine, and a local anesthetic, in amounts sufficient to protect orpreserve an organ.
 35. The composition of claim 34, wherein the localanesthetic is lignocaine.
 36. The composition of claim 34 wherein theorgan is a heart.
 37. A method for preserving or protecting an organcomprising contacting the organ with the composition of claim
 34. 38.The method of claim 37 wherein the composition is at a temperaturebetween about 15° C. to about 37° C.
 39. The method of claim 37 whereinthe organ is a heart.